Vaccination with a recombinant Saccharomyces cerevisiae expressing a tumor antigen breaks immune tolerance and elicits therapeutic antitumor responses. Saccharomyces cerevisiae, a nonpathogenic yeast, has been used previously as a vehicle to elicit immune responses to foreign antigens, and tumor-associated antigens, and has been shown to reduce tumor burden in mice. Studies were designed to determine if vaccination of human carcinoembryonic antigen (CEA)-transgenic (CEA-Tg) mice (where CEA is a self antigen) with a recombinant S. cerevisiae construct expressing human CEA (yeast-CEA) elicits CEA-specific T-cell responses and antitumor activity. CEA-Tg mice were vaccinated with yeast-CEA, and CD4+ and CD8+ T-cell responses were assessed after one and multiple administrations or vaccinations at multiple sites per administration. Antitumor activity was determined by tumor growth and overall survival in both pulmonary metastasis and subcutaneous (s.c.) pancreatic tumor models. These studies demonstrate that recombinant yeast can break tolerance and that (a) yeast-CEA constructs elicit both CEA-specific CD4+ and CD8+ T-cell responses; (b) repeated yeast-CEA administration causes increased antigen- specific T-cell responses after each vaccination; (c) vaccination with yeast-CEA at multiple sites induces a greater T-cell response than the same dose given at a single site; and (d) tumor-bearing mice vaccinated with yeast-CEA show a reduction in tumor burden and increased overall survival compared to mock-treated or control yeast-vaccinated mice in both pulmonary metastasis and s.c. pancreatic tumor models. Vaccination with a heat-killed recombinant yeast expressing the tumor-associated antigen CEA induces CEA-specific immune responses, reduces tumor burden, and extends overall survival in CEA-transgenic (CEA-Tg) mice. These studies thus form the rationale for the incorporation of recombinant yeast-CEA and other recombinant yeast constructs in cancer immunotherapy protocols.Studies comparing two or more vaccine platforms have historically evaluated each platform based on their ability to induce an immune response and may conclude that one vaccine is more efficacious than the other(s), leading to a recommendation for development of the more effective vaccine for clinical studies. Alternatively, these studies have documented the advantages of a diversified prime and boost regimen due to amplification of the antigen-specific T-cell population. We hypothesize here that two vaccine platforms targeting the same antigen might induce shared and distinct antigen-specific T-cell populations, and examined the possibility that two distinct vaccines could be used concomitantly. Using recombinant poxvirus and yeast vaccines, we compared the T-cell populations induced by these two platforms in terms of serum cytokine response, T-cell gene expression, T-cell receptor phenotype, antigen-specific cytokine expression, T-cell avidity, and T-cell antigen-specific tumor cell lysis. These studies demonstrated for the first time that vaccination with a recombinant poxvirus platform (rV/F-CEA/TRICOM) or a heat-killed yeast vaccine platform (yeast-CEA) elicits T-cell populations with both shared and unique phenotypic and functional characteristics. Furthermore, both the antigen and the vector play a role in the induction of distinct T-cell populations. We demonstrated that concurrent administration of two vaccines targeting the same antigen induces a more diverse T-cell population that leads to enhanced antitumor efficacy. These studies provide rationale for future clinical studies investigating concurrent administration of vaccine platforms targeting a single antigen to enhance antigen-specific immune response.